Functional Analysis Of Rice Heterotrimeric G-Protein γ Subunits RGG1 And RGG2

Rice (Oryza sativa) is the third highest world-wide production in agriculture commodity after sugarcane and maize. High yield in rice is the primary objective of plant breeding and has a vital importance for food security. Heterotrimeric G-proteins (α, β and y subunits) are involved in various signaling processes by transducing signals from an activated transmembrane GPCR to appropriate downstream effectors within cells. In plants, G proteins transmit signals by atypical mechanisms and effector proteins to control growth, cell proliferation, stomata opening, channel regulation, sugar sensing and some hormonal responses. It is well established that Gβγ dimers directly regulate K+-channels in mammals. The role of a and β subunits has been reported, also the role of Gy subunits (GS3 and qPE-9-1) has been reported but the function of Gy subunits (RGG1 and RGG2) has not been clearly stated. In this study rggl and rgg2 mutants were constructed using CRISPR/Cas9 system, this was done using pC1300 vector constructed from pCAMBIA1300 backbone. We also constructed RGG2 overexpressed using pZeroBack/Blunt vector. To create transgenic plants for this study, tissue culture was used and Agrobacterium mediated transformation was used for DNA transfer. In order to study the function of RGG2, we used RGG2 overexpressed line and Wuyunjing7 to treat in different concentrations of potassium for 0 day,6 hours,1 day,3 days,5 days and 7days. It was found that the target sequences of rggl and rgg2 were modified with insertions, substitutions and deletions. Phenotypic analysis of rggl and RGG2(OE) showed that transgenic plants were shorter than the wildtypes (Wuyunjing7 and Zhonghual 1). Even though RGG2(OE) is shorter than Wuyunjing7 it has more yield, but rggl has less yield than Zhonghual 1.When it came to the abiotic stress of K+-deficiency, Wuyunjing7 was more sensitive to K+-deficiency but RGG2(OE) was not. The results also show that RGG1 interacts with CNGC2 by bimolecular fluorescence complementation. Transcripts of some of the genes seemed to be upregulated by K+-starvation rather than down-regulated, which indicates that the expression level of some of the genes may have been restrained to slow down the growth rate of rice, while other genes were activated to adapt to long term K+deficiency. The expression of RGG2(OE) is highest on the 3rd day of deficiency. These findings indicate RGG2(OE) is induced by K+-deficiency.